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Original Articles |
Institute of Endocrinology (A.D.B., S.D.M., F.F., V.I.M., A.A.S.,
A.Be.) and
1 Department of Clinical and Experimental Medicine
"F.
Magrassi’ (G.F.A., D.G., A.Bi.), 2nd University of Naples, 80131
Naples, Italy
Address correspondence and requests for reprints to: Annamaria De Bellis, M.D., Istituto di Endocrinologia, Seconda Universita’ di Napoli, Via S. Pansini, 5, 80131 Napoli, Italy.
Abstract
It is commonly recognized that a few patients with Graves’ disease (GD) develop an overt ophthalmopathy, although most of them show subclinical extraocular muscle enlargement by appropriate imaging techniques. At present, it is not possible to identify the subgroup of GD patients with subclinical retroorbital connective involvement. Recently, it has been shown that increase of soluble intercellular adhesion molecule-1 (sICAM-1) serum levels is correlated to clinical activity score in active Graves’ ophthalmopathy (GO) patients with or without hyperthyroidism, suggesting that sICAM-1 serum values could reflect the degree of ocular inflammatory activity.
The aim of this longitudinal study was to evaluate sICAM-1 serum levels in GD patients without clinical ophthalmopathy and to assess their possible relationship with occurrence of GO. We measured sICAM-1 serum levels in 103 initially hyperthyroid GD patients without clinical ophthalmopathy and in 100 healthy subjects. All patients were treated with methimazole for 2 yr. Sera were collected from all patients before treatment and then monthly for the first 6 months of therapy, every 2 months in the following 6 months, and finally at the end of the follow-up study. Patients developing GO were excluded from the follow-up at the onset of ophthalmopathy. During the follow-up 17 GD patients (16.5%, group 1) developed overt eye involvement (14 as active inflammatory ophthalmopathy and 3 as ophthalmopathy without clinical retroorbital connective inflammation) and 86 (83.5%, group 2) did not. At start of the study, the mean of sICAM-1 serum concentrations did not differ significantly between the 2 groups, but it was significantly higher than in controls in both groups. No significant correlation between serum sICAM-1 concentrations and free thyroid hormone levels was found in the 2 groups of patients. During the follow-up study, a further increase of sICAM-1 serum levels was observed in 12 of the 14 patients (85.7%) of group 1 who developed active inflammatory ophthalmopathy not only at the onset but also before clinical GO appearance. On the contrary, the 3 patients of group 1 that developed ophthalmopathy without clinical retroorbital inflammation did not show any further increase of sICAM-1 levels at every time of follow-up in comparison with the starting values, even if their sICAM-1 levels were always higher than in normal controls. Finally, group 2 patients showed significantly decreased sICAM-1 levels throughout the follow-up period when compared with the starting values, although they were still significantly higher than in controls. These results indicate that a further increase of sICAM-1 serum levels before the onset of clinical ophthalmopathy may be a marker of subclinical retroorbital connective inflammation in GD patients. Therefore, our study suggests that serial determinations of sICAM-1 serum levels could help to identify and trace at the right time those GD patients prone to developing active inflammatory ophthalmopathy.
THYROID-associated ophthalmopathy (TAO) is an autoimmune process of the orbit involving both the extraocular muscles (EOM) and the retro-orbital connective tissue (1, 2, 3). T-lymphocytes and local release of cytokines have been shown to play an important role in the pathogenesis of retro-orbital connective tissue inflammation (4, 5, 6, 7). Moreover, it has been suggested that intercellular adhesion molecule 1 (ICAM-1) could be involved in crucial interactions among T-cells, macrophages, B-cells, endothelial cells, and retro-orbital fibroblasts (8, 9, 10, 11). Thus, in addition to surface ICAM-1 expression, increased levels of circulating form of this molecule (sICAM-1) have been found in sera of Graves’ disease (GD) patients with or without active ophthalmopathy (12, 13, 14).
We have recently found in active Graves’ ophthalmopathy (GO) patients with or without hyperthyroidism sICAM 1 serum levels significantly higher than those demonstrated in hyperthyroid GD patients without ophthalmopathy (15). Moreover, sICAM-1 levels were positively correlated to the clinical activity score of GO suggesting that these levels may be considered as a useful marker of retroocular endothelial cells and fibroblast activation in GD patients with clinical ophthalmopathy. However, sICAM-1 serum levels in GD patients with subclinical mild retroorbital connective tissue involvement have so far not been evaluated.
On this basis, this longitudinal study was aimed to evaluate the time course of sICAM-1 serum levels in hyperthyroid GD patients without GO and their possible relationship with the occurrence of ophthalmopathy.
Subjects and Methods
From May 1992 to September 1996, 119 patients with untreated hyperthyroid GD and no apparent ophthalmopathy consecutively entered the study. Patients suffering from infectious, allergic, or other autoimmune diseases were not included to avoid possible interferences with the sICAM-1 serum concentration. Orbital ultrasonography was performed in all patients: a normal pattern in 37 patients and a minimal EOM enlargement in the remaining 82 were evidenced.
Treatment and follow-up
All patients were treated with methimazole therapy for 24 months. Sixteen of 119 patients dropped out: 11 of them gave up the study while 5 became hypothyroid, thus requiring thyroxine supplements. The remaining 103 patients (73 females, 30 males, age 23–50) that reached a steady euthyroid state within two months of therapy completed the follow-up period successfully. All patients were examined for 2 yr, monthly in the first 6 months of therapy, then at 2 month-intervals in the following 6 months, and then at the end of the study. Because development of ophthalmopathy was considered as an end-point, the follow-up study was concluded when clinical ophthalmopathy occurred.
Diagnosis of ophthalmopathy was based on recent criteria recommended by the International Thyroid Association (16). Blood samples for sICAM-1 determinations were obtained from all patients at every examination during the study. In addition, in patients developing ophthalmopathy sICAM-1 serum levels were also evaluated at appearance of clinical eye involvement. As a control group, the sera of 100 healthy subjects were evaluated for sICAM-1 concentration only at beginning of the study.
Methods
Serum concentrations of sICAM-1 were measured by a sandwich ELISA method as previously described, using commercial kits from Bender MedSystem (Wien, Austria) (15).
Statistical analysis
Statistical evaluation of the difference of sICAM-1 values between the patient groups and the healthy controls was calculated by ANOVA. Frequencies were compared by the Fisher’s exact test. Soluble sICAM-1 levels were correlated, initially by the Spearman’s test, to free thyroid hormone concentrations in all patients, and subsequently to findings of eye involvement in patients developing ophthalmopathy.
Results
Patients were divided into 2 groups on the basis of ophthalmopathy appearance during the follow-up: 17 out of 103 patients (16.5%) developed clinical ophthalmopathy (group 1), while 86 patients (83.5%) did not (group 2).
Fourteen patients of group 1 showed ophthalmopathy characterized by
marked orbital connective tissue inflammation (clinical activity score
4); 8 of them also had EOM involvement. The remaining 3 patients of
group 1 had ophthalmopathy with predominant EOM involvement but without
clinical retroorbital connective tissue inflammation (activity
score = 0).
Clinical, demographic, and laboratory data of group 1 and group 2
patients at beginning of the study are summarized in Table 1
. No significant differences were
observed between the two groups of patients.
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During the follow-up period, 12 of the 14 patients in group 1 (85.7%)
that developed active ophthalmopathy showed, before overt eye
involvement and at the onset of clinical active inflammatory
ophthalmopathy, a further significant increase (100 ng/mL) of sICAM-1
serum levels with respect to their starting values (Fig. 1). Moreover, 7 showed, at the start of
the study, minimal EOM enlargement by orbital ultrasonography. The
remaining 2 patients not showing EOM enlargement at the beginning of
the study showed a further increase of sICAM-1 serum levels only at the
onset of active clinical GO.
|
). Moreover, in group 1 patients a positive
correlation of sICAM-1 serum levels with the activity score but not
with other findings of clinical ophthalmopathy was found at the onset
of ophthalmopathy. Finally, group 2 patients showed significant
decrease of sICAM-1 levels throughout the follow-up period when
compared with their starting values, although always significantly
higher than those observed in controls (Table 2).
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We have evaluated longitudinally the sICAM-1 serum levels in GD patients during a 2-yr follow-up period or until ophthalmopathy onset. The starting features of GD patients who developed ophthalmopathy were not significantly different from those observed in GD patients who did not develop ophthalmopathy. In particular, subclinical minimal EOM enlargement occurred in most patients in both groups, in accordance with several previous studies (17, 18, 19, 20). Interestingly, there was no difference in the prevalence of smokers between the two groups despite other studies suggesting cigarette smoking as a predisposing factor for GO (21, 22, 23); however, the small number of our selected patients suggests caution against our assumption. Initial sICAM-1 serum levels were similar in both groups of patients, in agreement with other studies (24, 25). During the follow-up-period, patients who did not develop ophthalmopathy showed a significant decrease of sICAM-1 serum levels in comparison with their starting values.
On the contrary, among GD patients who developed ophthalmopathy, 12 of
14 (85.7%) showed a further increase of sICAM-1 serum levels before
the onset of active inflammatory ophthalmopathy. These results suggest
that, in GD patients subsequently developing clinical active
ophthalmopathy, a marked orbital inflammation could be preceded by a
period of subclinical mild inflammation of retro-orbital tissues. The
origin of increased sICAM-1 levels in sera of these latter GD patients
is unknown. However, retroorbital tissues could be an important source
of serum sICAM-1, even if other potential sources like the thymus or
other lymphoid organs cannot be excluded (12). Because our GD patients
did not suffer from other autoimmune, allergic, and infectious
diseases, it is likely that subclinical and then clinically active
inflammatory ophthalmopathy could be responsible for increased sICAM-1
levels. Moreover, the decrease of sICAM-1 levels in patients who did
not develop GO excluded a significant role of the thyroid as source of
the further increase of sICAM-1 levels in patients who developed
ophthalmopathy. In accordance with our assumption, Heufelder and Bahn
(5, 9) showed, in marked inflamed retro-ocular connective tissue of GO,
both the presence of IFN
, TNF
, and IL1, and the elevated
expression of ICAM-1. Moreover, IFN, TNF, and IL1 have been
shown to strongly enhance surface expression of ICAM-1 in both
retroocular fibroblasts and orbital endothelial cells of GO (10, 11)
and to induce both improvement of periorbital inflammation and
reduction of sICAM-1 serum levels in corticosteroid and octreotide
therapy (12, 26). The increased sICAM-1 serum levels in sera of our
patients may simply be a consequence of proteolytic cleavage of surface
ICAM-1 expressed on activated fibroblasts and endothelial cells within
the mild inflamed orbit. On the other hand, they could be actively
secreted by these retroorbital cells, resulting from alternatively
spliced messenger RNA, which deletes the cytoplasmic and transmembrane
domains of the molecule in analogy to that demonstrated in other
inflamed tissues (27, 28). A circulating form of ICAM-1 in GO can play
a role in the ongoing immune process within the connective tissue in GO
(12), even if other factors can be involved (4, 8). In particular,
sICAM-1 may exert a cytokine-like signaling effect similar to the
costimulatory effect of surface bound adhesion molecules. In fact, GO
sera with high sICAM-1 serum levels markedly increase the peripheral
blood mononuclear cell adhesion to IFN-stimulated retroocular
fibroblast monolayers; furthermore this effect is hindered by sera with
anti-ICAM-1 MOAb (12). Thus, in GD patients a further significant
increase of sICAM-1 serum concentration could be considered an early
marker of subclinical mild retroocular inflammation in the
ophthalmopathy, predictive of the clinically marked retroocular
inflammation. In fact, in 3 GD patients who developed ophthalmopathy
without clinical connective inflammation, sICAM-1 concentrations,
although still higher than in controls, did not show further
significant increase. It has been demonstrated that, in orbital tissue
section from patients with severe active GO, ICAM-1 is not expressed in
extraocular muscle cells, being observed only in perimysial and
endomysial fibroblasts within extraocular muscles and in microvascular
endothelial cells, as well as in endothelial cell layer of larger blood
vessel and in fibroblasts throughout the connective tissue in the
posterior orbit (9). At the present time, in the literature, data on
surface ICAM-1 expression in GD patients without clinical active
inflammation are lacking. We suggest that in our 3 GD patients with
ophthalmopathy without clinical connective inflammation, surface ICAM-1
could be moderately expressed and circulating sICAM-1 could be shed or
secreted by the above mentioned cells in an amount capable of inducing
the increased basal sICAM 1 levels. Instead a further significant
increase occurs only when a more considerable amount of connective
tissue is involved, as found in the remaining patients of group 1
developing ophthalmopathy.
Thus our data suggest that serial determinations of sICAM-1 serum levels could help identify the subgroup of GD patients prone to develop severe clinical ophthalmopathy with retroocular connective inflammation. This could lead to development of new treatment strategies capable of hindering immune cell adhesion, with the ultimate goal of preventing clinical retroorbital endothelial and fibroblast activation in GO.
Received April 24, 1997.
Revised August 5, 1997.
Revised November 26, 1997.
Accepted December 10, 1997.
References
This article has been cited by other articles:
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  A. Kretowski, N. Wawrusiewicz, K. Mironczuk, J. Mysliwiec, M. Kretowska, and I. Kinalska Intercellular Adhesion Molecule 1 Gene Polymorphisms in Graves' Disease J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4945 - 4949. [Abstract] [Full Text] [PDF]
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